Consecutive treatments of methamphetamine promote the development of cardiac pathological symptoms in zebrafish

Chronic methamphetamine use, a widespread drug epidemic, has been associated with cardiac morphological and electrical remodeling, leading to the development of numerous cardiovascular diseases. While methamphetamine has been documented to induce arrhythmia, most results originate from clinical trials from users who experienced different durations of methamphetamine abuse, providing no documentation on the use of methamphetamine in standardized settings. Additionally, the underlying molecular mechanism on how methamphetamine affects the cardiovascular system remains elusive. A relationship was sought between cardiotoxicity and arrhythmia with associated methamphetamine abuse in zebrafish to identify and to understand the adverse cardiac symptoms associated with methamphetamine. Zebrafish were first treated with methamphetamine 3 times a week over a 2-week duration. Immediately after treatment, zebrafish underwent electrocardiogram (ECG) measurement using an in-house developed acquisition system for electrophysiological analysis. Subsequent analyses of cAMP expression and Ca2+ regulation in zebrafish cardiomyocytes were conducted. cAMP is vital to development of myocardial fibrosis and arrhythmia, prominent symptoms in the development of cardiovascular diseases. Ca2+ dysregulation is also a factor in inducing arrhythmias. During the first week of treatment, zebrafish that were administered with methamphetamine displayed a decrease in heart rate, which persisted throughout the second week and remained significantly lower than the heart rate of untreated fish. Results also indicate an increased heart rate variability during the early stage of treatment followed by a decrease in the late stage for methamphetamine-treated fish over the duration of the experiment, suggesting a biphasic response to methamphetamine exposure. Methamphetamine-treated fish also exhibited reduced QTc intervals throughout the experiment. Results from the cAMP and Ca2+ assays demonstrate that cAMP was upregulated and Ca2+ was dysregulated in response to methamphetamine treatment. Collagenic assays indicated significant fibrotic response to methamphetamine treatment. These results provide potential insight into the role of methamphetamine in the development of fibrosis and arrhythmia due to downstream effectors of cAMP.

Author response: Thank you for the valuable feedback!We have conducted additional experiments, including the production of TAAR1-overexpressed HEK293 cells for cAMP and Ca 2+ analysis.We have also utilized EPPTB, a selective TAAR1 antagonist, as an inhibitor to illustrate the integral role of TAAR1 in downstream dysregulation.The new data can be found in Fig 3.
Line 432-433: "The results in this study also indicated that the upregulation of cAMP occurred in a dose-dependent manner and can be inhibited by an antagonist, displaying the plausible reversibility of Meth-induced cardiotoxicity".This conclusion cannot be drawn from the presented results.
Author response: Thank you for the comment!The statement has been modified to "Using the GloSensor cAMP assay, we demonstrated that the upregulation of cAMP occurred in a dosedependent manner within zebrafish cardiomyocytes.This upregulation was inhibited by an antagonist of cAMP."Notably, we removed the statement on the reversibility on Meth-induced cardiotoxicity.
(2) Experimental Design and Interpretation.Another important problem is the apparently arbitrary experimental design, including the lack of appropriate controls.The authors are treating animals with Meth, and then measuring defects in electrical activity and cAMP, but the characterization is incredibly superficial.Specific examples: • Is this set-up standard to do electrophysiological recording?Why the need to come up with their own?
Author response: Thank you for the questions!The use of micro-needle electrodes for zebrafish ECG recording was first developed in the PI's previous lab [1].Our lab continues on this line of research to improve the set-up for various cardiovascular applications.The set-up described in the current manuscript was designed to facilitate easier and faster manipulation of the electrodes.This feature was vital for conducting ECG measurements for this study and future drug testing studies in a feasible manner.
• Please justify treatment concentrations and durations: please add justification on the dose of Meth used: why 200 µm?Why 20 min of treatment at 200 µm?Why 3 times a week?Does that correlate somehow with drug intake in humans?Why quantification only for two weeks?
Author response: Thank you for your questions!The treatment regimen of twenty minutes and the concentration for Meth were first determined from a previous zebrafish Meth study [2].Prior to the study described by our manuscript, we conducted a pilot study to determine the suitable concentration for reducing mortality and maximizing significance.We proceeded with 200 μM as the experimental concentration.We treated the zebrafish 3 times a week in order to allow the zebrafish at least one day to recover from measurement, as tricaine, the anesthetic agent, has been known to affect ECG parameters due to its effect to block sodium channels [3].As there were no previous studies analyzing Meth-associated cardiotoxicity in zebrafish, we hope that the study detailed in our manuscript will provide a basis for future Meth studies.The duration of two weeks was initially set based on previous long-term Meth-associated cardiotoxicity studies [4,5].
• Figure 5: HEK293 used as control to test for cell specificity.I am not sure this is the most appropriate control.Perhaps a better one to claim that meth specifically induces cardiotoxicity would have been to isolate other cells types from treated fish and do the same quantification.
Author response: Thank you for the suggestion!While we have considered using other cell types as a control for cAMP expression, we are currently not able to isolate those cell types in a reliable manner.As an alternative, we developed a HEK cell line with overexpressed zebrafish TAAR1 as a positive control.
• Not clear how the dose-dependent experiments have been performed in Figure 5C-D: same cells subjected to different increasing doses, or different cells?
Author response: Thank you for the question!The original cell population isolated from the zebrafish heart was separated into the respective groups for treatment with different increasing doses.
1) Rigor and Reproducibility of the Data.
• In figure 4: what would be the explanation for the drop of HR also in untreated fish at day 8 compared to other days (which looks significant to other days in untreated fish)?
Author response: Thank you for the question!The explanation for the drop of HR between days 5 and 8 is the consistent exposure to tricaine.As an anesthetic agent, tricaine has been known to decrease muscle contractility and heart rate [6].Repeated tricaine treatment for zebrafish induced increased susceptibility to the effects of tricaine, as shown in a previous study [7].This susceptibility may be varied between individual organisms.As shown in the second iteration of our experiment, the heart rate in untreated fish did not display a noticeable decline throughout the course of the experiment.However, the conclusion of our study delineates that Meth-treated fish exhibited a greater decrease in heart rate than untreated fish, accounting for tricaine effects for both experimental groups.
• Figure 4B: For this quantification, it looks like the HRV is actually also increasing after 10 days in untreated fish.Please comment.
Author response: Thank you for the insight!The slow increase in HRV in untreated fish near the conclusion of the experiment may be due to the presence of arrhythmias due to tricaine treatment, which increased the variation in heart rate.It has been established that anesthetic agents cause arrhythmic instances [8].In the second trial, we determined that HRV did not increase in untreated fish.The conclusion drawn from our results indicates that Meth induces additional arrhythmias during the first stage of our study, followed by a decrease in HRV due to the significant decrease in heart rate overtaking the presence of arrhythmias.
• Figure 4C: For this result, it is worth noting that based on the quantification, it actually looks like the PR stayed pretty constant in Meth-treated fish (~55ms), and that the differences between the two experimental groups seem to actually be carried by variations of the PR in the untreated ones along the course of the experiment (ranging from ~55 up to 69 ms).Explanations?
Author response: The variation in the PR interval may be due to the effects of anesthesia, as the agent slows heart conduction.In our second trial, we determined that PR interval did not significantly change between the untreated and treated groups, and the discussion has been modified accordingly.We would like to emphasize that the major conclusions of this manuscript include the progressive decrease in heart rate and the differences seen in HRV.The differences in PR interval were determined to be significant only in the early stage of treatment, and the explanation for this observation is most likely the antagonistic effects of anesthesia and Meth.Author response: Thanks for pointing this out!The aforementioned comments were addressed in the respective figures (Figs 2 and 3) and in the 'GloSensor cAMP assay' subsection under 'Methods'.We have replaced forskolin with TAAR1-overexpressed HEK293 cells as the positive control.

MINOR COMMENTS:
Not clear why the potential effect of tricaine is being brought up in the discussion when the authors do not address that at all experimentally.
Author response: Thank you for the comment!We realized that we did not fully clarify the potential confounding effects of tricaine (anesthetic agent) on the ECG data we have presented in the study.
In this manuscript the authors describe a preparation and a study in which they record cardiac activity from adult zebrafish following methamphetamine treatment.The goal of the work was to examine the relationship between cardiotoxicity and arrhythmia with associated methamphetamine exposure in adult zebrafish.Animals were treated with Meth 3 times a week for 2 weeks and multiple ECG measurements were taken.The authors also examined cAMP levels via a cAMP assay to ascertain the effect on this second messenger following Meth treatment.I am reasonably convinced that meth acts either directly or indirectly on cardiac activity, but I am more skeptical about the mechanism and the nature of the interactions.The authors have not provided convincing evidence that zebrafish either have TAAR1 and that it is expressed in cardiac tissue (PCR or immunohistochemistry if antibodies exist).I understand that antagonists for TAAR1 are limited but a compound such as EPPTB which (I believe) is an inverse agonist might provide some information on the actions of Meth on TAAR1 in this study.I have also found a number of grammatical and spelling errors throughout the manuscript and respectfully suggest that the authors pay particular attention to these details.
Additional comments are listed below.
1. Abstract, Line 31-32: the sentence that reads "zebrafish underwent electrocardiogram (ECG)…" needs to be adjusted because many readers may not know what the previously developed acquisition system is.In other words, clarify what the authors are referring to.
Author response: Thank you for pointing this out!The sentence was revised to "…zebrafish underwent electrocardiogram (ECG) measurement using an in-house developed acquisition system for electrophysiological analysis."This clarifies that the system is based on previous work conducted in our lab, which is referenced in the introduction.
2. Abstract, line 36-38: Should probably read "…exhibited a decrease in heart rate at the end of the first week of treatment, which remained significantly lower than untreated fish in the second week.
6. Methods, line 169: This sentence sounds a bit odd.Please review and correct if necessary.
3. Introduction, line 97-98: It may be how I read the sentence but to me this sentence implies that all GPCRs use cAMP as a second messenger.Please adjust for accuracy.
12. Figure 5: Methamphetamine, Forskolin and Arrhythmia are misspelled.Please double check all spelling.
Author response: Thank you for the comments.We have revised the corresponding sentences for accuracy and clarity.

4.
What is known about TAAR1 in zebrafish?can the authors provide evidence (maybe a reference or PCR etc) for TAAR1 expression in zebrafish?
Author response: Thank you for your question!Research regarding TAAR1 expression in zebrafish has mainly been conducted for the neurological system, specifically, the olfactory system [9].Similar neurological studies in other animal models have been published.However, the understanding of how TAAR1 expression may influence cardiovascular pathology remains poorly understood, even though TAAR1 has been discovered to be expressed in the heart [10].Given that Meth triggers cAMP via TAAR1 expression, it would be beneficial to explore Meth-induced TAAR1 expression and modulation for future cardiovascular studies.We have produced TAAR1overexpressed HEK293 cells to demonstrate that Meth upregulates TAAR1 and causes downstream dysregulation, as seen in Fig 3 .5. Methods, line 155: I think this is the first time PDMS is used.Please explain what this is.
Author response: Thank you for the question!PDMS, or polydimethylsiloxane, is a flexible and biocompatible polymer most commonly used in biosensors and implants.Therefore, it serves as a suitable material for housing zebrafish.

Figure 1 -
Figure 1-3 should be merged.They have only two panels each (A,B).Poorly integrated and discussed in the text.Some conclusions only described in figure legends.

7 .
Methods, line 219: Please explain what is BDM?Author response: Thank you for asking!BDM, or butanedione monoxime, is a contraction inhibitor for cardiomyocytes.Used by Sander et al. for zebrafish cardiomyocyte isolation, BDM was most likely utilized to increase viability of the cardiomyocytes [11].8. Methods line 241: Please explain what is the JMP suite?Author response: Thanks for asking!The JMP suite is a statistical analysis and data visualization software.9. Results: I found little indication of numbers of experiments and animals throughout the Results section or in the Figure legends.Author response: Thank you for the comment!We have included the number of animals used (control, n=6; treatment, n=8) as well as the number of trials (2) in the relevant sections in Results and Figures.10.
Acknowledgements: It seems odd to me to Acknowledge persons who are already listed as Authors of the manuscript.Author response: Thank you for pointing this out.The corresponding section has been removed.11. Figure 4: Apologies if I missed this but can the authors Discuss why there are some effects at intermediate time periods (eg Fig 4C Day 5) but not at earlier or later time periods (Day 3 or Day 8, 10).